The genome sequence of the Brown Long-eared bat, Plecotus auritus (Linnaeus 1758)

We present a genome assembly from a female Plecotus auritus (Brown Long-eared bat; Chordata; Mammalia; Chiroptera; Vespertilionidae). The genome sequence is 2163.2 megabases in span. Most of the assembly is scaffolded into 16 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.91 kilobases in length.


Background
The Brown Long-eared bat, Plecotus auritus (Linnaeus 1758), is a small insectivorous bat with slow hovering flight found in and near woodland and hedgerows.Although able to echolocate it seems mainly to use its extremely long ears to listen for prey, and its relatively large eyes, to catch prey such as moths.It also gleans insects and spiders from leaves and bark.It can fold its ears down when at rest (Swift, 1988).
Plecotus auritus is noted as of Least Concern on the IUCN Red List (IUCN, 2022).It is found across Europe, from the UK in the west, to the Urals in the east.In the UK it regularly roosts in lofts of houses and has a small home range.Because of its slow hovering flight and preference for house lofts and gleaning, it is vulnerable to capture by cats and to being caught in sticky insect traps (Swift, 1988).
The genome of the Brown Long-eared bat, Plecotus auritus, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomal-level genome sequence for Plecotus auritus, based on a euthanised female specimen from East Sussex, UK.

Genome sequence report
The genome was sequenced from a female Plecotus auritus (Figure 1) collected from Ringmer, East Sussex, England (50.89, 0.05).A total of 34-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 33 missing joins or mis-joins, reducing the scaffold number by 21.69%, and increasing the scaffold N50 by 4.10%.
The final assembly has a total length of 2,163.2Mb in 64 sequence scaffolds with a scaffold N50 of 186.5 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.84%) of the assembly sequence was assigned to 16 chromosomallevel scaffolds, representing 15 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosome X was assigned based on synteny to Pipistrellus pygmaeus (GCA_949987585.1)(Ruedi et al., 2023).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A female Plecotus auritus (specimen ID NHMUK014438751, ToLID mPleAur1) was collected from Ringmer, East Sussex, England, UK (latitude 50.89, longitude 0.05) on 2021-09-25.This specimen was caught by a cat on 4 September 2021 in Ringmer, East Sussex BN8 5HZ and brought to the Hurstpierpoint bat hospital, where its condition was found to be life threatening, with one or more open wounds.Initially feeding, given antibiotics and painkillers, it became weaker, went off its food and developed breathing difficulties.On 25 September it was deemed kindest to euthanise, which was carried out by cervical dislocation.The animal was immediately dissected and its tissue was preserved by freezing.The specimen was collected and identified by Amanda Millar (Sussex Bat Group) and tissues were stored at -80 °C.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the mPleAur1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the muscles and organs was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).
HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop

Evaluation of final assembly
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al., 2017) DSL2 Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the

Software tool Version
of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Michael R Buchalski
California Department of Fish and Wildlife, Sacramento, CA, USA In this manuscript A. Millar and collaborators present a chromosome-length de novo genome assembly for Plecotus auritus.In my opinion, the sequencing and assembly methods are comparable to other high-quality assemblies for other non-model organisms, short of telomereto-telomere assembly.My major comment is that the manuscript would benefit from the addition of basic information on genus/species biology, methodology, and results.I have not reviewed this journal before, so I'm not familiar with your format and philosophy.This manuscript is marked as a 'Data Note'.From what I found online, there are no stated word/length limits for this type of article.Therefore, I would imagine such information could be added without violating manuscript length limitations.
A good example of my point is the abstract.I could find no limitations on abstracts other than 300 words max.So why is this abstract ~50 words to the exclusion of so much important and relevant information?More specifically, what is the geographic range and listing status of P. auritus?What are the characteristics of the genus that make it interesting for genomic investigation?What were the scaffold and contig N50 values for the assembly, or BUSCO scores?Why is this assembly a valuable contribution to bat research?
The remainder of the manuscript follows this trend of absolute minimalism, where I can find no such requirements in your guidelines to authors for Data Notes.The manuscript would be a more effective read if the authors did not repeatedly refer to internal laboratory protocols published online when methods could be adequately described in a few sentences.And it is insufficient to repeatedly claim that various laboratory kits were used 'following manufacturer recommendations'.I refer the authors to the several articles describing genome assemblies conducted by the California Conservation Genomics Project published in Journal of Heredity.Dozens of articles implementing similar methods can be found that strike an excellent balance of brevity and detail.As written, it is difficult to fully evaluate what the authors have done.

Xiuguang Mao
East China Normal University, Shanghai, China Millar et al. present a high-quality chromosome-scale assembly for Plecotus auritus and the total number of chromosomal-level scaffolds corresponds to the haploid chromosome number of this species (2n=32).This genome provides valuable resource to study the karyotype evolution in Vespertilionidae.
One minor comment or suggestion: In the background section, if the authors can provide more specific and special characteristics about Plecotus auritus (e.g.forage, root selection, echolocation and karyotype), it will be better for readers to understand the application of this reference genome.

Is the rationale for creating the dataset(s) clearly described? Partly
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: evolutionary biology, speciation, comparative genomics, genome assembly, bats I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Is the rationale for creating the dataset(s) clearly described? No
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Figure 2 .Figure 3 .
Figure 2. Genome assembly of Plecotus auritus, mPleAur1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 2,163,221,563 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (248,793,850 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (186,501,726 and 62,501,937 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the laurasiatheria_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUOHF01/dataset/CAUOHF01/snail.

Figure 4 .
Figure 4. Genome assembly of Plecotus auritus, mPleAur1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAUOHF01/dataset/CAUOHF01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Plecotus auritus, mPleAur1.1:Hi-C contact map of the mPleAur1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=BwOAeHE5S6eID4YvhIko0Q.

Table 1 . Genome data for Plecotus auritus, mPleAur1.1. Project accession data
Rhie et al. (2021)benchmarks are adapted from column VGP-2020 of "Table1: Proposed standards and metrics for defining genome assembly quality" fromRhie et al. (2021).RNA was extracted from tissue of mPleAur1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Dentonetal., 2023b).

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature

Table 3 . Software tools: versions and sources.
The genome sequence is released openly for reuse.The Plecotus auritus genome sequencing initiative is part of the Darwin Tree of Life (DToL) project.All raw sequence data and the assembly have been deposited in INSDC databases.The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute.Raw data and assembly accession identifiers are reported in Table1.

the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Partly Competing Interests:
No competing interests were disclosed.
Specific comments can be found below:No Are

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 21 June 2024 https://doi.org/10.21956/wellcomeopenres.23772.r86495© 2024 Mao X.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.